The long-term objective of this project is the elucidation and molecular understanding of the cascade of reactions that couples the synaptic receptor and channel on retinal depolarizing bipolar cells (DBCs). The postsynaptic action of glutamate, the transmitter that is released onto DBCs, is unusual for an excitatory amino because it produces a hyperpolarization by closing synaptic channels. these channels are believed to be gated by a diffusible intracellular messenger. Our specific aims are to identify the intracellular messenger that opens the synaptic channel in DBCs of tiger salamander, and to determine the mechanism by which glutamate controls the levels of this intracellular messenger. We also wish to learn if Ca2+ can modulate any step in the cascade and what consequence such modulation might have for synaptic transmission between photoreceptors and DBCs. (I) The candidacy of cGMP as the intracellular messenger will be accessed. We will attempt to measure the ability of cyclic nucleotides (cAMP and cGMP) to open directly synaptic channels by applying cyclic nucleotides to patches excised from DBCs. The ion selectivity and cyclic nucleotide affinity of the channels will be determined. We will also attempt to impose jumps in the intracellular concentration of cGMP and measure the response. Inactive "caged" cGMP will be dialyzed into DBCs and then activated by flash photolysis. (II) Glutamate is thought to lower the levels of cGMP by activating a cAMP-phosphodiesterase (PDE). We will test this hypothesis by dialyzing poorly hydrolyzed analogs of cGMP into intact DBCs. If hydrolysis of cGMP by PDE is an obligatory step in the cascade, then these analogs should prevent glutamate from closing channels. (III) We will use both excised patches and intact isolated cells to measure the permeability of Ca2+ through the glutamate channels. We will also monitor the levels of Ca2+ in intact cells with fura-2 while recording responses to glutamate in order to determine whether Ca2+ levels change during glutamate stimulation. (IV) We will measure the effects of changing Ca2+ levels on the synaptic current. DBCs will be dialyzed with one of two different "caged" Ca2+ chelators. One chelator binds Ca2+ after uncaging, and the other binds Ca2+ prior to uncaging. The chelators will be uncaged by light and their effects on the synaptic current will be accessed.